PROJECT SUMMARY The use of current and emerging genetically encoded tools could greatly benefit from advanced methods for gene delivery to the desired cell population. When used in conjunction with transgenic animals to restrict expression to cell populations of interest, adeno-associated viruses (AAVs) can provide well-tolerated and targeted transgene expression that enables long-term behavioral, in vivo imaging, and physiological experiments. Lacking from the current suite of vector tools is a way to achieve cell- or circuit-specificity with AAVs without the use of transgenic animals. We pioneered a powerful strategy that allows for the generation and selection of viral vectors with optimized properties by Cre-recombination-dependent AAV targeted evolution (CREATE). We have used CREATE to evolve AAVs that are capable of crossing the blood?brain barrier (BBB) and transducing most cells in the adult brain. These systemically delivered AAVs enable noninvasive CNS-wide transduction of specific cell types and regions in rodents when used with gene regulatory elements. We propose to build upon our success with the CREATE method to develop a suite of systemic viral vector tools and approaches that will enable cell-state monitoring in defined cell populations/circuits and noninvasive modulation of complex behaviors in wild-type animals. Methods for noninvasive modulation of specific circuits need to couple actuators gated by highly penetrant moieties (BBB-permeant ligands, ultrasound, etc) with noninvasive delivery methods that have a brain-wide reach, yet can confine actuator expression to specific circuits. The systemic AAVs we developed provide a solution to the latter. We will: develop AAV genomes for expression of genetically encoded calcium indicators in specific cell types without transgenesis (Aim 1); engineer AAV capsids capable of anterograde trans-synaptic trafficking (Aim 2); and provide validation for noninvasive control of behavior in transgenic and wild-type animals (Aim 3). These novel vector reagents and protocols will be distributed, upon validation, to the neuroscience research community by our established resource center (www.clover.caltech.edu). We expect that these vector reagents will provide new avenues for studying and ultimately treating the vertebrate nervous system. Potential uses for the systemic viruses are: circuit mapping; fast screening of gene regulatory elements; and genome editing with CRISPR-Cas9. In addition, when paired with appropriate activity modulator genes, the new AAVs could enable noninvasive deep-brain modulation. Importantly, the broadly efficient and mosaic expression strategies developed and validated in this project will be compatible with other sensors and actuators developed for circuit studies, even, long-term, across species.